Follicle stimulating hormone stimulation of 125-I-human chorionic gonadotropin binding in porcine granulosa cell cultures.

In order to see if FSH acts directly upon the granulosa cell to stimulate hCG binding, granulosa cells harvested from small 1-2 mm porcine follicles were grown in 250 ml flasks in chemically defined media containing 0.05 mug/ml highly purified human FSH for 2, 4, and 6 days. The defined medium consisted of culture medium 199 plus 0.4% bovine serum albumin, 0.2% lactalbumin hydrolysate and 10 munit/ml insulin. The cultures were harvested by scraping with a rubber policeman and incubated with 0.1 mug/ml 131-I- or 125-I-hCG. Binding expressed as cpm/culture or per mg protein yielded similar results. In five separate experiments addition of FSH stimulated hCG binding two- to fourfold above control cultures. In a typical experiment after 2 days of culture, the specific binding of control cultures to hCG was 962 plus or minus 45 cpm/culture (-x plus or minus SE; n = 3) and the binding in cultures grown in the presence of 0.05 mug/ml FSH was 3933 plus or minus 1787 (n = 3; P less than 0.01). Granulosa cells harvested from large (8-12 mm) follicles grown under similar conditions bound 29,669 plus or minus 948 cpm/culture (n = 4). These data demonstrate that FSH may have a direct stimulatory role upon induction of granulosa cell LH-hCG receptors in vitro.     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Follicle-stimulating hormone plays a role in the induction of ovarian follicular cysts in hypophysectomized rats.

Unabated stimulation by low doses of LH-like activity produces ovarian follicular cysts in both progesterone-synchronized immature rats and pregnant rats. Serum FSH is maintained in both of these models at values similar to those observed on diestrus. To determine whether unabated stimulation by basal serum FSH affects the ability of LH-like activity to induce cystic ovaries, immature hypophysectomized (HYPOXD) rats were given either no hormone (control); 2 micrograms ovine FSH (oFSH) once daily for 14 days beginning on Day 27; 0.5 IU hCG twice daily for 13 days beginning on Day 28 of age; or both oFSH and hCG (FSH + hCG) beginning on Day 27 and Day 28, respectively. By the end of the in vivo treatments (Day 40 of age), the largest follicles in the ovaries of control and hCG-treated HYPOXD rats were at the preantral stage of development, whereas the largest follicles present in ovaries from FSH-treated animals were atretic and at the small antral stage of development. In contrast, ovaries from rats treated with FSH + hCG displayed large follicular cysts by Day 37 of age. Of the serum steroids analyzed, only estradiol and androstenedione concentrations for animals treated with FSH + hCG were consistently elevated above values observed for control HYPOXD rats. Serum testosterone and dihydrotestosterone values were similar for hCG-treated and control HYPOXD rats throughout the in vivo treatments. In contrast, these steroids were elevated between Days 3 and 5 of FSH treatment (+/- hCG treatment). Serum progesterone and estrone values for all in vivo gonadotropin treatment groups were similar to those of controls. Serum insulin concentrations were not affected by any in vivo treatment. Incubates of follicles/cysts from FSH + hCG-treated HYPOXD rats contained more progesterone, androstenedione, and estradiol than incubates of follicles from any other in vivo treatment group. Follicles from all in vivo treatment groups responded to 8-bromo cAMP (cAMP) with increased in vitro progesterone accumulation. However, only follicles from FSH-treated and FSH + hCG-treated rats responded to cAMP with increased androstenedione and estradiol accumulation in vitro. Inclusion of 400 ng of either androstenedione or testosterone in the incubation medium enhanced progesterone accumulation in follicular incubates from control, hCG-treated, and FSH-treated HYPOXD rats, but did not enhance progesterone accumulation in follicular incubates from FSH + hCG-treated animals. Both androstenedione and estradiol production increased markedly under these conditions for follicles from all in vivo treatment groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Formation of estrogen glucuronides by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization

The formation of steroid glucuronides by human granulosa cells isolated from human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG)-stimulated cycles for in vitro fertilization was studied. From granulosa cells in suspension, 5 x 10(-7) M androstenedione was converted into estradiol (2.50 +/- 0.21 ng/ml), estrone (1.84 +/- 0.16 ng/ml), estradiol glucuronide (0.38 +/- 0.07 ng/ml), as well as estrone glucuronide (0.24 +/- 0.04 ng/ml). When 5 x 10(-7) M estradiol was incubated, estrone (15.5 +/- 0.9 ng/ml) and estradiol glucuronide (0.12 +/- 0.05 ng/ml) were detected in medium. Using the same preparation of granulosa cells, we have observed that androsterone could uniquely be transformed into androstane-3 alpha, 17 beta-diol (1.42 +/- 0.56 ng/ml), and only low amounts of steroid glucuronides could be detected. Since the formation of steroid glucuronides was extremely small when granulosa cells in suspension were used, we subsequently studied granulosa cells in culture. When 5 x 10(-7) M estradiol was added, estrone (7.8 +/- 1.3 ng/ml) and estradiol glucuronide (0.68 +/- 0.08 ng/ml) were formed. The addition of follicle-stimulating hormone did not cause a further increase in estrone or estradiol glucuronide levels. As observed with granulosa cells in suspension, incubation with androsterone led to the formation of androstane-3 alpha, 17 beta-diol (24.2 +/- 0.07 ng/ml). Our data demonstrated the presence of glucuronyltransferase in human granulosa cells obtained from preovulatory follicles of hMG/hCG-treated women. In addition, since the conversion of androsterone into C-19 steroid glucoronide was relatively small, the present finding also indicates that the glucoronyltransferase enzymatic activity in granulosa-luteal cells preferentially conjugated estrogens.     

Effect of follicle size on in vitro production of steroids and insulin-like growth factor (IGF)-I,IGF-II,and the IGF-binding proteins by equine ovarian granulosa cells

Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medium (16-25 mm), and large (25-48 mm) follicles were collected from cyclic mares (n = 14), cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells.     

Secretion of cumulus expansion-enabling factor (CEEF) in porcine follicles

The objective of this study was to find out whether porcine cumulus and mural granulosa cells can secrete cumulus expansion-enabling factor (CEEF). Culture drops of M-199 medium were conditioned with denuded porcine oocytes (1 oocyte/μl), cumulus cells from oocytectomized complexes (1 OOX/μl), pieces of mural granulosa isolated from preantral to preovulatory follicles (1000 cells/μl), or oviductal cells (1000 cells/μl) for 24 hr. The production of CEEF was assessed by the addition of mouse OOX and follicle-stimulating hormone (FSH) (1 μg/ml) to microdrops of the conditioned medium. After 16–18 hr, expansion of the mouse OOX was scored on a scale of 0 to 4 by morphologic criteria. Mouse OOX did not expand in nonconditioned FSH-supplemented medium. Immature porcine oocytes produced +3 to +4 expansion of the mouse OOX. Granulosa cells isolated from preantral and early antral follicles and cumulus cells isolated from all stages of follicle development constitutively secreted CEEF under in vitro conditions. Mural granulosa cells of small, medium, and preovulatory (PMSG) follicles also secreted CEEF in vitro; however, FSH or leutenizing hormone (LH) stimulation was essential for this secretion. Hormonally induced secretion of CEEF was accompanied by expansion of the mural granulosa itself. Granulosa cells isolated from follicles of gilts 20 hr after PMSG and human chorionic gonadotropin (hCG) administration did not produce CEEF and did not expand in response to FSH and LH in vitro. CEEF activity also was found in the follicular fluid of small antral follicles, was reduced in medium follicles, and was not detectable in PMSG-stimulated follicles. However, CEEF activity was reestablished in the follicular fluid of preovulatory follicles by hCG injection, conceivably due to increased production of CEEF by cumulus cells. We conclude that (1) porcine cumulus and mural granulosa cells are capable of CEEF production in vitro and (2) autocrine secretion of CEEF by cumulus cells is involved in regulation of porcine cumulus expansion both in vitro and in vivo. Mol. Reprod. Dev. 49:141–149, 1998. © 1998 Wiley-Liss, Inc.     

Effects of fetal bovine serum, FSH and 17beta-estradiol on the culture of bovine preantral follicles

We describe a 7-d culture in droplets of collagen gel of isolated small bovine preantral follicles in medium with or without 10% fetal bovine serum (FBS). In addition, the effect of human recombinant FSH and 17beta-estradiol on the morphology and growth of the preantral follicles was investigated in medium without FBS. After culture in medium with 10% FBS, the increase in follicle diameter was 13.1 +/- 8.4 microm, the percentage of BrdU-labeled cells was 49.9 +/- 11.3 and the number of cells per area granulosa was 11.1 +/- 1.8. Omission of serum from the culture medium had no effect on the percentage of labeled cells, but the diameter increase was lower and the cells were smaller. Apparently, serum affects the size of the granulosa cells from small preantral follicles rather than the stimulation of cell proliferation. Addition of human recombinant FSH and/or 17beta-estradiol to serum-free medium resulted in a larger diameter increase during culture compared with that of the control. With FSH, this was due to an increase in cell proliferation, while with estradiol this was caused by an increase in granulosa cell size. The effects of simultaneous treatment with FSH and estradiol was simply the combination of their individual effects. In conclusion, small bovine preantral follicles can be cultured for 7 d in the absence of serum and hormones. The follicles increase in diameter and react to FSH with enhanced cell proliferation and to estradiol with an increase in cell size.     

FSH induced stimulation of catalase activity in goat granulosa cells in vitro

Reactive oxygen species scavenging enzymes like catalase play diverse role in mammals. The presence of catalase in mammalian ovary is now well established. In the present investigation, changes in catalase activity in granulosa cells isolated from follicles at various stages of differentiation in response to FSH were studied. The follicles were dissected out from goat ovaries and classified as small (<3 mm), medium (3–6 mm) or large (>6 mm). Granulosa cells were isolated from categorized follicles. Results showed that there was a three-fold increase in catalase activity in granulosa cells from large follicles as compared to small and medium follicles. The catalase activity was stimulated significantly when granulosa cells were treated with FSH in vitro. The minimum effective dose that could stimulate catalase activity and estradiol secretion in case of granulosa cells from small and medium sized follicles was 100 ng/ml; for larger follicles, this value was 200 ng/ml. Concomitant to the increase in catalase activity, the estradiol secretion was significantly enhanced when cultured goat granulosa cells were treated with FSH. It was concluded that enzyme catalase may have a functional role in goat ovarian follicular development under endocrine regulation.     

Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles.

During the development of oocyte-granulosa cell complexes from preantral follicles in vitro, oocytes grow and acquire competence to undergo germinal vesicle breakdown (GVB). In the culture system used here, GVB-competent oocytes were maintained in meiotic arrest solely by endogenous physiological mechanisms of the granulosa cells without supplementation with meiosis-arresting substances. Addition of mycophenolic acid, an inhibitor of inosine monophosphate (IMP) dehydrogenase, induced GVB in about 70% of the GVB-competent oocytes grown in vitro. The mechanism for meiotic arrest in this system is, therefore, similar to that for arrest in vivo insofar as it requires the participation of the IMP dehydrogenase pathway. Rp-cyclic adenosine monophosphothioate, a membrane-permeable antagonist to cAMP, induced GVB by about 30% of the competent oocytes. Cyclic AMP-dependent pathways, therefore, participate in the physiological mechanism by which mouse granulosa cells maintain meiotic arrest. Complexes were grown for 10 days in medium containing 0, 1, 5, or 10 ng/ml FSH, were stimulated with either 1 microgram/ml FSH or LH, and were assessed for GVB and cumulus expansion. GVB was stimulated by FSH whether or not the complexes were grown in medium containing FSH, but LH or hCG induced GVB only when the complexes were grown in medium containing FSH. Cumulus expansion occurred in response to either FSH or LH only when complexes were grown in medium containing FSH. FSH, therefore, promotes the differentiation of granulosa cells from preantral follicles in vitro so that LH can stimulate GVB and cumulus expansion.     

Hormonal regulation of ovarian cellular proliferation

The steroid hormone estradiol, and the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are known to be essential for the growth and differentiation of follicles in the ovary. The present study was conducted to determine quantitatively the effects of estradiol, FSH and LH on proliferation of different ovarian cell types (granulosa and theca cells). The immature female hypophysectomized rate sequentially primed with estradiol, FSH and LH was used as the experimental model. Proliferation was assessed by examining changes in total DNA, incorporation of 3H-thymidine into DNA and labeling index in specific cell types. Estradiol and FSH each acted on follicles at different stages of development to stimulate proliferative activity of both granulosa and theca cells. Continued administration of either hormone caused a decrease in the proliferative activity of both cell types. These observations have been interpreted to indicate that estradiol and FSH can each alter the length of the specific phases of the cell cycle. A luteinizing dose of LH caused a cessation of proliferation in luteinizing granulosa cells while stimulating a limited proliferation of theca cells. Absence of the appropriate hormonal stimulus caused both granulosa and theca cells to stop proliferating and the follicles to undergo atresia. These results indicate that, depending upon the state of differentiation of granulosa and theca cells, estradiol, FSH and LH can stimulate or inhibit the ability of these cells to proliferate.     

Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy

Alterations in the basal lamina (BL) of developing follicles were studied by immunofluorescent microscopy using antibodies against type IV collagen, laminin, and fibronectin, and by electron microscopy. Ovarian development was induced in immature rats by sequential administration of estradiol, follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). A continuous BL was observed in healthy follicles treated with estradiol and FSH. As determined by immunofluorescence, laminin, type IV collagen, and fibronectin were restricted to the BL and the theca but not to the granulosa. When follicles were allowed to undergo atresia or induced to ovulate with hCG, the BL became fragmented. This was confirmed by electron microscopy of healthy, atretic, and luteinizing follicles which showed that in healthy follicles the BL was continuous, whereas in both atretic and luteinizing follicles, it was fragmented. Atresia was also associated with the penetration of thecal cells into the follicles. These observations indicate that the intact BL present in healthy follicles undergo extensive changes during atresia and ovulation.     

Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles

The purpose of this study was to establish a culture model for isolated intact porcine antral follicles and investigate the relationship between granulosa cell apoptosis and follicular atresia. Small (<3 mm), medium (3–5 mm) and large (>5 mm) healthy porcine follicles were isolated and cultured in serum‐free TCM199 with or without follicular stimulating hormone (FSH). Microscopic identification of healthy follicles was confirmed by histology. A spontaneous onset of apoptotic cell death in granulosa cells was observed from cultured antral follicles. The apoptotic rate of granulosa cells from small follicles cultured for 24 hr was higher than those of large and medium follicles, accompanied with high FasL mRNA abundance in granulosa cells. Supplementation with 3 or 5 IU/ml FSH significantly inhibited the percentage of granulosa cells that became apoptotic. FSH did not significantly alter estradiol secretion from cultured follicles. Progesterone secretion significantly decreased after culture for 48 hr, coinciding with the morphological changes observed. FasL and Fas mRNA were expressed in the healthy, early atretic, and progressed atretic porcine follicles regardless of follicular size. However, FasL but not Fas mRNA levels increased during follicular atresia. Addition of FSH significantly decreased FasL rather than Fas mRNA levels in granulosa cells and could attenuate apoptosis. Small follicles seemed to be more susceptible to atresia as compared to medium and large follicles. Mol. Reprod. Dev. 77: 670–678, 2010. © 2010 Wiley‐Liss, Inc.     

Prolactin modulates steroidogenesis by rat granulosa cells: II. Effects on estradiol

The effects of prolactin on secretion of estradiol by rat granulosa cells obtained at two stages of differentiation and cultured under various conditions were determined. Relatively undifferentiated granulosa cells were obtained from immature, diethylstilbestrol-treated, hypophysectomized (HPX) rats and cultured in serum-free medium or with 10% serum. More highly differentiated granulosa cells were obtained on the morning of proestrous from the preovulatory follicles of immature rats in which an estrous cycle had been induced with 4 IU pregnant mare's serum gonadotropin; these cells were cultured in medium containing 10% serum. Cells were cultured for 3 days with graded doses of prolactin (0, 0.02, 0.2, 2, or 10 micrograms/ml) alone or in combination with follicle-stimulating hormone (FSH; 300 ng/ml), testosterone (0.5 microM), or FSH + testosterone. In control cultures (no prolactin) the relatively undifferentiated granulosa cells from HPX rats secreted negligible quantities of estradiol except when both FSH and testosterone were supplied. Prolactin alone or in combination with FSH or testosterone had no effect on estradiol secretion, but prolactin in combination with FSH + testosterone significantly decreased secretion in a dose-dependent fashion. This set of prolactin treatments was applied in both serum-free medium and medium containing 10% serum, with similar results under both culture conditions. The inhibitory effects of prolactin appeared to be reversible if cells were cultured with prolactin for only 1 day, but were not reversed if cells were cultured with prolactin for 2 or 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH-stimulated follicular secretions enhance oocyte maturation in pigs

Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theca and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 mug/ml FSH (FSH-stimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2.5mug/ml FSH, 2.5 mug/ml LH and 20 ng/ml PRL, FSH-LH-PRL-stimulated conditioned medium; 54.1 vs 28.5%; P=0.001). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=0.06). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=0.65), but were higher than those of oocytes matured in fresh control medium (P<0.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

A granulosa cell differentiation-stage-specific cytotoxic activity in bovine and rat sera

Heat-inactivated serum is cytotoxic to granulosa cells from preantral follicles but not to cells from preovulatory follicles. A dominant feature of the granulosa cells of preovulatory follicles is the presence of luteinizing hormone (LH) receptors on the surface of the cells. In the present study, we have examined the relationship between the process of LH receptor induction and the acquisition of serum tolerance in granulosa cells in vitro. Granulosa cells from the ovaries of immature rats primed with diethylstilbestrol (DES) were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium containing 30 ng of ovine follicle-stimulating hormone (oFSH; NIH-15). At either 0, 24, or 48 h of culture, heat-inactivated fetal bovine serum (FBS) was added (10% by volume) to separate groups of culture tubes. All cells were cultured for a total of 72 h, at which time the cultures were assessed for LH receptor (specific 125I-human chorionic gonadotropin [hCG] binding) and DNA content. LH receptors were induced in all FSH-containing serum-free cultures by 48 h. Receptors were not induced, however, when serum was added after either 0 or 24 h of culture. Furthermore, serum addition at these times resulted in a cell loss (assessed by DNA) of 40-60%. Serum addition at 48 h to FSH-containing cultures resulted in an inability to detect LH receptors at 72 h and with no significant effect on the culture DNA content. Addition of a protein extract of FBS at the initiation of cell culture prevented FSH-stimulated LH receptor induction and was cytotoxic. A lipid extract of FSH did not interfere with receptor induction and was not cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

Covalent transfer of heavy chains of inter-alpha-trypsin inhibitor family proteins to hyaluronan in in vivo and in vitro expanded porcine oocyte-cumulus complexes

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with IalphaI antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

Granulosa cell steroidogenesis before in vitro fertilization

Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband's sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 microM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with "over-ripeness" and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.